Folate, B12 and one-carbon metabolism

Folate forms

Humans must obtain folate from food ¹. Cells use it to hold single carbon atoms for making DNA bases and the amino acid methionine ¹.

Folic acid, the synthetic form added to foods and supplements, becomes active only after dihydrofolate reductase (DHFR) reduces it to tetrahydrofolate (THF) ²¹.

Thymidylate and purines

Dividing cells use single carbon atoms to make the DNA base thymine and the purine bases ¹.

Thymidylate synthase transfers the carbon of 5,10-methylene-THF to dUMP, forming dTMP and dihydrofolate (DHF) ¹.

Thymidylate cycleCytosol, nucleus and mitochondria

Tetrahydrofolate (THF)5,10-Methylene-THFSerine hydroxymethyltransferase (SHMT1, SHMT2)L-Serine → glycine, Pyridoxal 5′-phosphate
5,10-Methylene-THFDihydrofolate (DHF)Thymidylate synthase (TYMS)dUMP → dTMPFluorouracilPemetrexedFolate supplies the carbon and the two electrons that reduce it to methyl
Dihydrofolate (DHF)Tetrahydrofolate (THF)Dihydrofolate reductase (DHFR)NADPHMethotrexateAlso reduces folic acid to DHF, slowly in human liver

Methotrexate blocks DHFR and traps folate as DHF ¹.

MTHFR and the 677C>T variant

The enzyme MTHFR converts part of the folate pool into methyl-folate, which supplies methyl groups for methionine ³. A common variant of its gene, 677C>T, makes the enzyme less stable ³.

Cell extracts from TT homozygotes have about 30% of the activity of CC extracts ³. About 10–15% of people of European origin and of Japanese are TT homozygotes ³. The variant is the most frequent genetic cause of mild hyperhomocysteinaemia ³.

Methionine synthase and vitamin B12

Methionine synthase requires vitamin B12 ⁴. It moves the methyl group from methyl-folate to homocysteine, which regenerates methionine and frees folate ⁴².

Folate cycle, methyl branchCytosol

Tetrahydrofolate (THF)5,10-Methylene-THFSerine hydroxymethyltransferase 1 (SHMT1)L-Serine → glycine, Pyridoxal 5′-phosphateSHMT2 catalyses the same reaction in mitochondria
5,10-Methylene-THF5-Methyl-THFMethylenetetrahydrofolate reductase (MTHFR)NADPH, FADIrreversible in vivo; inhibited by SAM; Ala222Val (677C>T) loses FAD faster
5-Methyl-THFTetrahydrofolate (THF)Methionine synthase (MTR)Homocysteine → methionine, MethylcobalaminNitrous oxideOnly exit from 5-methyl-THF; slowed in B12 deficiency (methyl-folate trap)

When B12 is lacking, methyl-folate accumulates, because no other reaction consumes it and the MTHFR reaction does not reverse ². This methyl-folate trap produces a functional folate deficiency ²⁵.

S-adenosylmethionine and homocysteine

Methionine is converted into S-adenosylmethionine, the main donor of methyl groups ⁶. Each methyl transfer leaves homocysteine, which is converted back to methionine or broken down to cysteine ⁶.

Folic acid reduction and the folate one-carbon cycleCytosol of hepatocytes and dividing cells

Folic acid
Dihydrofolate reductase (DHFR)NADPHMethotrexateSlow and variable in human liver; reduced food folates bypass this step
7,8-Dihydrofolate
Dihydrofolate reductase (DHFR)NADPHMethotrexateAlso reduces the dihydrofolate released by thymidylate synthase (TYMS)
Tetrahydrofolate
Serine hydroxymethyltransferase (SHMT1 cytosolic, SHMT2 mitochondrial)Serine to glycine, Pyridoxal 5′-phosphateBranch: TYMS transfers this carbon to dUMP, forming dTMP for DNA
5,10-Methylenetetrahydrofolate
Methylenetetrahydrofolate reductase (MTHFR)NADPH, FADIrreversible in vivo; inhibited by SAM; the 677C>T variant lowers activity
5-Methyltetrahydrofolate
Methionine synthase (MTR)Methylcobalamin, Homocysteine to methionineVitamin B12 deficiency slows this step and traps folate as 5-methyl-THF
Tetrahydrofolate

In 15 randomised trials with 71,422 participants followed for 1 to 7.3 years, lowering homocysteine with vitamins B6, B9 or B12 did not prevent myocardial infarction ⁷. It occurred in 7.1% of treated and 6.0% of placebo participants (risk ratio 1.02, 95% CI 0.95 to 1.10) ⁷. Stroke occurred in 4.3% and 5.1% (0.90, 0.82 to 0.99), a crude absolute difference of 0.8 percentage points and a number needed to treat of about 125 ⁷.

Megaloblastic anaemia

When folate or B12 is lacking, the bone marrow cannot make DNA properly, and the blood contains too few red cells, which are larger than normal ¹².

Low 5,10-methylene-THF slows thymidylate synthesis, so DNA polymerase incorporates uracil ⁵. In folate-deficient adults (n = 8), blood DNA contained eight times as many uracils as in controls (n = 14) ⁵. B12 deficiency can also cause a myeloneuropathy through demyelination, which is almost never seen in folate deficiency ².

References

Independent research
Ducker GS, Rabinowitz JD. One-Carbon Metabolism in Health and Disease Cell Metabolism, 2017.
doi:10.1016/j.cmet.2016.08.009 PubMed 27641100
Funding and finding
Funding
G.S.D. is supported by a postdoctoral fellowship from the American Cancer Society (PF-15-190-01-TBE). J.D.R. is supported by NIH grants R01CA163591 and P30DK019525 and Stand Up to Cancer.
Finding
Review. Animals require dietary folate. Tetrahydrofolate (THF) binds one-carbon units at N5 and N10 at three oxidation states: 5-methyl-THF, the most reduced, 5,10-methylene-THF and 10-formyl-THF, the most oxidised; folic acid is reduced to dihydrofolate (DHF) and then THF. SHMT1 (cytosol) and SHMT2 (mitochondria) interconvert serine and glycine with THF and 5,10-methylene-THF, reversibly; in rapidly proliferating cultured cells the main direct source of cytosolic one-carbon units is formate released from mitochondria, reassimilated by the trifunctional MTHFD1. TYMS methylates dUMP to dTMP with 5,10-methylene-THF and is the only folate enzyme that alters THF itself, taking two electrons from it and producing DHF; DHFR regenerates THF with NADPH and is the target of trimethoprim and methotrexate, which trap folate as DHF. Purine synthesis incorporates two 10-formyl-THF units; in proliferating cells each new DNA or RNA base needs on average one 10-formyl-THF, and 5,10-methylene-THF is needed for one in four DNA bases. 5-Fluorouracil is a suicide inhibitor of TYMS; methotrexate most potently blocks DHFR, whereas pemetrexed more potently inhibits TYMS and AICAR transformylase; 5-formyl-THF (folinic acid, leucovorin) is given as rescue. 5-Methyl-THF, made by cytosolic NADPH-dependent MTHFR, is consumed only by cobalamin-dependent methionine synthase; BHMT is limited to liver and kidney. SAM inhibits MTHFR and activates CBS; 5-methyl-THF inhibits GNMT. Transsulfuration is the only route that degrades homocysteine. Folate and cobalamin supplementation lower homocysteine without reducing cardiovascular events in the general population. Dietary folate deficiency in adults manifests as macrocytic anaemia.
Regulator
EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA Panel), Turck D, Bohn T, Castenmiller J, de Henauw S, Hirsch-Ernst KI, et al. Scientific opinion on the tolerable upper intake level for folate EFSA Journal, 2023.
doi:10.2903/j.efsa.2023.8353 PubMed 37965303
Funding and finding
Funding
European Food Safety Authority, on request from the European Commission (question EFSA-Q-2021-00366). Preparatory systematic reviews were conducted by a contractor, Karolinska Institutet in collaboration with the University of Oslo, under an EFSA procurement procedure.
Finding
Folates consist of a pterin moiety linked to p-aminobenzoic acid and one or more glutamates, substituted at N5 or N10 by one-carbon units; natural folates are reduced polyglutamates usually with five to seven glutamates. A jejunal hydrolase (EC 3.4.17.21) converts polyglutamates to monoglutamates, which PCFT (SLC46A1), the most important intestinal transporter, and RFC (SLC19A1) move into enterocytes; 5-methyl-THF is the main circulating form; RFC, the main transporter from blood to tissues, has about 100 times lower affinity for folic acid than for reduced folates. Folylpolyglutamate synthase retains folates as THF polyglutamates; 5-methyl-THF monoglutamate and folic acid are poor substrates, and 5-methyl-THF is lost from cells unless methionine synthase converts it to THF. Folic acid, a fully oxidised monoglutamate, is reduced by DHFR via dihydrofolate to THF. The MTHFR reaction is essentially irreversible under physiological conditions and SAM is its allosteric inhibitor; in cobalamin deficiency 5-methyl-THF is metabolically trapped, SAM falls, MTHFR becomes fully active, and a functional folate deficiency develops with raised plasma 5-methyl-THF and homocysteine, depleted erythrocyte folate and fewer methyl groups for transmethylation. The megaloblastic anaemia of cobalamin deficiency is identical to that of folate deficiency; neurological manifestations (myeloneuropathy from demyelination) can occur with or without anaemia, are almost never seen in folate deficiency, and have an unknown mechanism, with congenital defects of MTHFR and methionine synthase pointing to methionine synthase rather than methylmalonyl-CoA mutase. Folic acid can initiate DNA synthesis in cobalamin deficiency through DHFR but cannot correct the methylation defect. MTHFR 677TT is associated with lower serum folate and higher homocysteine; in a genome-wide study of 2232 Irish adults aged 18 to 28 years MTHFR 677C>T was the only significant modifier of red cell and serum folate. ULs for folic acid retained: 1000 ug/day for adults including pregnant and lactating women, with progression of neurological symptoms in cobalamin-deficient patients as critical effect.
Independent research
Yamada K, Chen Z, Rozen R, Matthews RG. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase Proceedings of the National Academy of Sciences of the United States of America, 2001.
doi:10.1073/pnas.261469998 PubMed 11742092
Funding and finding
Funding
These studies were supported in part by National Institutes of Health Grant GM24908 (to R.G.M.) and by funds from the Medical Research Council of Canada (to R.R.).
Finding
Human MTHFR is a dimer of about 70 kDa subunits, each with non-covalently bound FAD, that reduces 5,10-methylene-THF to 5-methyl-THF with NADPH; SAM binds the C-terminal regulatory domain as an allosteric inhibitor, and SAH competes with SAM there without inhibiting. Purified recombinant Ala222Val enzyme (677C>T) had the same Km for NADPH and 5,10-methylene-THF, the same Vmax and the same SAM inhibition as wild type, but released FAD about 3-fold faster after dilution to 50 nM subunits at 37 degrees C; 5-methyl-THF and SAM slowed FAD loss. Background cited: cell extracts from TT homozygotes have about 30% of CC activity; about 10-15% of Caucasians of European origin and of Japanese are TT, more in Mediterranean countries and fewer among African Americans; 677C>T is the most frequent genetic cause of mild hyperhomocysteinaemia, seen in TT homozygotes with plasma folate below the median.
Independent research
Gherasim C, Lofgren M, Banerjee R. Navigating the B12 Road: Assimilation, Delivery, and Disorders of Cobalamin Journal of Biological Chemistry, 2013.
doi:10.1074/jbc.R113.458810 PubMed 23539619
Funding and finding
Funding
This work was supported, in whole or in part, by National Institutes of Health Grant DK45776.
Finding
Review. Mammals have two B12-dependent enzymes: methionine synthase, which uses methylcobalamin, and methylmalonyl-CoA mutase, which uses adenosylcobalamin to convert methylmalonyl-CoA to succinyl-CoA. Methionine synthase works in two half-reactions: methylcobalamin methylates homocysteine to methionine, leaving cob(I)alamin, which removes the methyl group from 5-methyl-THF to give THF and methylcobalamin; occasional oxidation to cob(II)alamin is repaired by reductive methylation needing NADPH, methionine synthase reductase and S-adenosylmethionine. Food cobalamin binds haptocorrin; in the duodenum pancreatic proteases release it, intrinsic factor binds it, and intestinal cells take it up by cubilin/AMN receptor-mediated endocytosis. About 20% of circulating cobalamin is bound to transcobalamin, which delivers it to cells through the transcobalamin receptor. Mutations in the cblG (methionine synthase) and cblE (reductase) loci cause isolated hyperhomocysteinaemia without methylmalonic aciduria.
Independent research
Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage Proceedings of the National Academy of Sciences of the United States of America, 1997.
doi:10.1073/pnas.94.7.3290 PubMed 9096386
Funding and finding
Funding
This work was supported by National Cancer Institute Outstanding Investigator Grant CA39910, by National Institute of Environmental Health Sciences Center Grant ESO1896 (B.N.A.), and by the intramural programs of the National Institute of Environmental Health Sciences and National Health and Environmental Effects Research Laboratory (R.B.E.). B.C.B. thanks the Biomedical Mass Spectrometry Unit at the University of New South Wales for financial support during the writing of a portion of the article.
Finding
Folate-deficient adults (erythrocyte folate below 140 ng/ml, n = 8) had a geometric mean of 3,960,000 uracils per diploid cell in blood DNA, 8-fold more than controls (498,000, n = 14; P = 0.003); after 5 mg folic acid daily, measured at 6 to 8 weeks, the value fell to 186,000 (n = 5; P = 0.0003). Mechanism proposed: low 5,10-methylene-THF decreases dTMP synthesis, raises the dUMP/dTMP ratio and leads to DNA polymerase-mediated dUTP misincorporation; excision by uracil-DNA glycosylase and apyrimidinic endonuclease creates transient nicks, and two opposing nicks can form a double-strand break. Vitamin B12 deficiency traps folate as 5-methyl-THF, causing functional folate deficiency, homocysteine accumulation and uracil misincorporation.
Independent research
Lu SC, Mato JM. S-adenosylmethionine in liver health, injury, and cancer Physiological Reviews, 2012.
doi:10.1152/physrev.00047.2011 PubMed 23073625
Funding and finding
Funding
This work was supported by National Institutes of Health Grants R01DK51719, R01DK092407 and P30DK48522 (to S. C. Lu), R01AT1576 and R01AT004896 (to S. C. Lu and J. M. Mato) as well as Plan Nacional of I+D SAF 2011-29851 and Departamento de Educación Gobierno Vasco 2011 (to J. M. Mato).
Finding
Review. Methionine adenosyltransferase synthesises S-adenosylmethionine (SAM) from methionine and ATP; tripolyphosphate formed in the reaction is hydrolysed to orthophosphate and pyrophosphate before product release. MAT1A is largely expressed in normal liver and MAT2A in all extrahepatic tissues. Over 200 human proteins are known or putative SAM-dependent methyltransferases; GNMT is the most abundant in liver (1% of cytosolic protein); the liver carries out up to 85% of all methylation reactions. S-adenosylhomocysteine (SAH) inhibits methyltransferases with a Ki in the micromolar range of their Km for SAM. AHCY cleaves SAH reversibly into adenosine and homocysteine; the thermodynamics favour SAH synthesis, and hydrolysis proceeds only if adenosine and homocysteine are rapidly removed. Homocysteine is remethylated by methionine synthase, expressed in all tissues, or by BHMT, limited to liver and kidney, with betaine; transsulfuration converts it to cysteine through CBS and cystathionase, both requiring vitamin B6. SAM inhibits MTHFR and activates CBS.
Independent research
Martí-Carvajal AJ, Solà I, Lathyris D, Dayer M. Homocysteine-lowering interventions for preventing cardiovascular events Cochrane Database of Systematic Reviews, 2017.
doi:10.1002/14651858.CD006612.pub5 PubMed 28816346
Funding and finding
Participants
71,422
Follow-up
1 to 7.3 years
Funding
Internal sources: no sources of support provided. External sources: Iberoamerican Cochrane Centre, Spain (academic); Cochrane Heart Group, UK (academic).
Finding
15 RCTs, 71,422 participants, follow-up 1 to 7.3 years. Against placebo, homocysteine-lowering interventions showed no difference in myocardial infarction (7.1% against 6.0%; RR 1.02, 95% CI 0.95-1.10; 12 trials, 46,699 participants), death from any cause (11.7% against 12.3%; RR 1.01, 0.96-1.06; 11 trials, 44,817) or serious adverse events (8.3% against 8.5%; RR 1.07, 1.00-1.14), and were associated with reduced stroke (4.3% against 5.1%; RR 0.90, 0.82-0.99; 10 trials, 44,224). Background: normal total homocysteine is close to 10 umol/L; fasting 12-30 umol/L is termed mild or moderate, 31-100 intermediate and above 100 severe hyperhomocysteinaemia.
Industry-funded
Aroda VR, Edelstein SL, Goldberg RB, Knowler WC, Marcovina SM, Orchard TJ, et al. Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study Journal of Clinical Endocrinology and Metabolism, 2016.
doi:10.1210/jc.2015-3754 PubMed 26900641
Funding and finding
Participants
1,800
Follow-up
mean 5 and 13 years
Funding
The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health is the primary funding agency, with further funding from other NIH institutes, the Indian Health Service, the Department of Veterans Affairs, the Centers for Disease Control and Prevention and the American Diabetes Association. Bristol-Myers Squibb and Parke-Davis provided additional funding and material support during the DPP, Lipha (Merck-Santé) provided medication, and LifeScan Inc donated materials during the DPP and DPPOS.
In-kind support
Medication from Lipha (Merck-Santé); materials from LifeScan Inc.
Finding
Serum B12 at or below 203 pg/mL at a mean 5 years: 37 of 859 (4.3%) on metformin against 20 of 856 (2.3%) on placebo, P = 0.02; at a mean 13 years: 56 of 753 (7.4%) against 40 of 736 (5.4%), P = 0.12. B12 at or below 298 pg/mL: 19.1% against 9.5% at 5 years and 20.3% against 15.6% at 13 years. Odds ratio for B12 deficiency per year of metformin use 1.13 (95% CI 1.06 to 1.20).
Regulator
EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA). Scientific Opinion on Dietary Reference Values for cobalamin (vitamin B12) EFSA Journal, 2015.
doi:10.2903/j.efsa.2015.4150 PubMed 42109795
Funding and finding
Funding
European Food Safety Authority, on request from the European Commission (question EFSA-Q-2011-01227); preparatory work by the EFSA Working Group on Dietary Reference Values for vitamins and EFSA staff.
Finding
A combination of biomarkers of cobalamin status, namely serum cobalamin, holotranscobalamin, methylmalonic acid and plasma total homocysteine, was judged the most suitable basis for Dietary Reference Values; an Average Requirement could not be determined. Adequate Intake for adults 4 ug/day, based on biomarker data and observed mean intakes of 4.2 to 8.6 ug/day in adults in several EU countries; 4.5 ug/day in pregnancy and 5 ug/day in lactation; 1.5 ug/day at 7 to 11 months rising to 4 ug/day at 15 to 17 years.
Independent research
Clarke R, Bennett DA, Parish S, Verhoef P, Dötsch-Klerk M, Lathrop M, et al. Homocysteine and coronary heart disease: meta-analysis of MTHFR case-control studies, avoiding publication bias PLoS Medicine, 2012.
doi:10.1371/journal.pmed.1001177 PubMed 22363213
Funding and finding
Participants
116,136
Funding
Supported by grants from the British Heart Foundation and UK Medical Research Council to the University of Oxford Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU), and the Oxford BHF Centre for Research Excellence (JCH). No funding bodies played any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In-kind support
Unpublished genotype datasets contributed by collaborating groups, among them deCODE (Reykjavik), whose dataset was contributed by an employee co-author.
Finding
In 19 unpublished datasets (48,175 CHD cases, 67,961 controls), the MTHFR 677TT against CC genotype gave a CHD odds ratio of 1.02 (95% CI 0.98-1.07), and 1.01 (0.95-1.07) in unsupplemented low-folate populations; homocysteine was expected to be about 20% higher with TT than CC. In 86 published studies (28,617 cases, 41,857 controls), the odds ratio was 1.15 (1.09-1.21), significantly discrepant (p = 0.001); the authors conclude that the discrepancy reflects publication bias or methodological problems.
Industry-funded
Holmes MV, Newcombe P, Hubacek JA, Sofat R, Ricketts SL, Cooper J, et al. Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk: a meta-analysis of genetic studies and randomised trials Lancet, 2011.
doi:10.1016/S0140-6736(11)60872-6 PubMed 21803414
Funding and finding
Funding
Public and charitable funders listed in the Acknowledgments, among them the UK Medical Research Council, British Heart Foundation, Wellcome Trust, UK Department of Health, US National Institute of Neurological Disorders and Stroke, US National Institute on Aging, National Health and Medical Research Council of Australia, National Center for Geriatrics and Gerontology of Japan, Erasmus MC and Dutch ministries, the Netherlands Heart Foundation, the Danish Medical Research Council and Danish Heart Foundation. The funding sources had no role in study design, data collection, analysis, interpretation, writing or the decision to submit.
Finding
The TT against CC difference in homocysteine was 3.12 umol/L (95% CI 2.23 to 4.01) in Asia and 0.13 umol/L (-0.85 to 1.11) in high-folate America, Australia and New Zealand; the stroke odds ratios were 1.68 (1.44 to 1.97) and 1.03 (0.84 to 1.25). In trials of homocysteine-lowering interventions the summary stroke RR was 0.94 (0.85 to 1.04); no trial had evaluated homocysteine lowering exclusively in a low-folate region. Classed industry because employees of GlaxoSmithKline took part in the analyses.

Updated 2026-10-21